Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extreme acid conditions

Brucella is an expanding genus of major zoonotic pathogens including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock and humans. Recently, we have shown that in the new species Brucella microti the glutamate decarboxylase (GAD)-dependent system contributes to survival at a pH of 2.5 and also to infection in mice by oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates representative of all known species and strains of this genus and 16 strains of the closest neighbor genus Ochrobactrum were studied using microbiological, biochemical and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2 and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid-resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the E. coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD-negative.